The role of jnk1 during zebrafish development

Abstract

The c-Jun N-terminal Kinases 1-3 (JNK1-3) are mitogen activated protein kinases (MAPK) involved in the non-canonical Wnt / planar cell polarity (PCP) signalling pathway. In mouse models and human patients, mutations in the PCP pathway have been associated with congenital heart malformation; however investigating the function of Jnk using null mice has been difficult because of genetic redundancy and embryonic death that occurs in Jnk1/Jnk2 compound mutants. Both human and mouse JNK1 orthologs have four known transcripts which differ in sequence at two locations. Although evidence has been published to show that these transcripts have differential downstream binding affinities, no transcript-specific functions have been suggested. Zebrafish possess two jnk1 paralogs on chromosomes 12 (jnk1b) and 13 (jnk1a) that arose from the teleost genome duplication event. The aim of this thesis was to identify the jnk1 transcripts that arose from the zebrafish jnk1 genes and compare them to what is seen in human JNK1. Furthermore I aimed to knock-down zebrafish jnk1 translation during development using morpholino oligonucleotides (MOs) and assess the phenotypes caused. Considering the involvement of PCP in cardiac development, I hypothesised that jnk1 would be required for cardiac morphogenesis, and that some subfunctionalization would exist between zebrafish jnk1a and jnk1b paralogs which would explain the retention of both duplicated genes. I was able to clone four different transcripts from each of the jnk1 paralogs, and these transcripts closely resembled mouse and human orthologs. Semi-quantitative RT-PCR demonstrated that these transcripts had differential expression levels during development and in adult tissues. When knocked down, jnk1a and jnk1b morphants were viable (to 72hpf) but displayed multiple developmental defects that were paralog-specific. The jnk1a morphants displayed retinal layer underdevelopment, structural immaturity of the heart and uncoupling of the jog-loop cardiac morphogenetic movements. In contrast the jnk1b morphants displayed a high frequency of reversed cardiac situs with a milder overall heart phenotype, in addition to curled body axis formation and failure to form the somitic horizontal myoseptum. Dual knockdown resulted in a combination of paralog-specific defects, as well as developmental delay and a failure for the heart tube to loop. These results demonstrated that the human and zebrafish JNK1 genes are highly conserved II and suggested that following genome duplication the zebrafish jnk1 paralogs have undergone subfunctionalization. The jnk1a gene appears to have organ specific roles and in particular is required for heart growth whereas the jnk1b gene appears to be important for somatic left-right signalling and therefore heart looping and jogging. This work expands on what is known about the two jnk1 paralogs and strengthens their validity as a model of human JNK1. Establishment of a zebrafish knockdown model provides a powerful tool for the investigation of jnk1 in an organism that has many advantages for developmental biology research. The demonstration that several organs require jnk1 during development will allow for future work to investigate the exact role of this gene during organogenesis, and the involvement in left-right axis patterning strengthens the evidence that JNK acts in the PCP pathway.